Since the discovery of hybridoma technology in 1975 and with the subsequent development of recombinant DNA technologies, the genes encoding binding proteins, particularly antibodies, have been engineered to manipulate the binding protein or antibody in some way, e.g., reduction in size, such as a single-chain variable fragment (scFv) or variable fragments from antibody light chain (VL) and heavy chain (VH); rebuilding into multivalent high-avidity reagents; and fusion with a variety of molecules (e.g. enzymes, toxins). In addition, several selection methods and experimental approaches have been developed in order to select monoclonal antibodies (mAbs) with higher affinity and specificity.
Antibody selection techniques using an antibody library can be performed totally in vitro, without the requirement of cell transformation, or in vivo, in which cells may be transformed, e.g., using vectors encoding an antibody library. In vitro antibody selection includes techniques such as ribosome, RNA and DNA display, while in vivo antibody selection includes techniques such as phage-display, two-hybrid systems, cell-display, e.g., in bacteria, yeast, or mammalian cells and protein fragment complementation assays (PCA). Phage-display is broadly used due to its simplicity, versatility and ability to be adapted to many specific conditions. Yeast, bacterial, and mammalian cell display platforms offer an advantage over phage-display in that fluorescence activated cell sorting (FACS) can be coupled with cell surface antibody display to allow monitoring of both antibody expression on the cell surface and the ability of that antibody to bind to its target. On the other hand, ribosomal display technology allows the screening of larger libraries and facilitates the diversity and efficient antibody maturation in vitro.
Although a large number of antibodies have been selected by various technologies, most need to be affinity improved or enhanced. Such improvement in antibody affinity and subsequent selection of specific antibody are typically achieved through the use of one or more methods, systems, or technologies, such as multimerization of antibodies; E. coli mutator strains; B-cell lines, e.g., RAMOS, DT-40, or HEK293T cells and activation-induced cytidine deaminase (AID); retrovirus display systems; error-prone PCR; immunoglobulin chain or CDR shuffling; directed mutagenesis.
Despite the various methodologies developed to improve the affinity and subsequent selection of antibodies, significant problems are prevalent. Examples of problems that have been detected and reported include potency/affinity, expression, correct folding and post-translation modifications of isolated antibodies. Thus improved and new processes are needed for selecting specific and functional antibodies and optimizing them, as well as other types of binding molecules, particularly for therapeutic utility. It is therefore important to develop new strategies related to improved and useful methods for introducing diversification into the genes or nucleic acid sequences encoding binding molecules, such as antibodies, and to provide novel and efficient methods for selecting the expressed molecules, particularly for generating new, improved and useful therapeutic biological products.